The human T-cell leukemia viruses (HTLV) represent a family of T-cell retroviruses with three known members. HTLV type I (HTLV-I) has transforming activity in vitro and is etiologically linked to adult T-cell leukemia, which is known to be endemic in several parts of the world. HTLV-II is another retrovirus having transforming capacity in vitro, and has been isolated from a patient with a T-cell variant of hairy cell leukemia (for a review of HTLV-I and II see Cann and Chen). HTLV-III, which has also been called lymphadenopathy-associated virus and is now known as the human immunodeficiency virus (HIV), is lytic for certain kinds of T cells and has been linked to the etiology of acquired immunodeficiency syndrome (AIDS). Unlike the HTLV-I and -II viruses, HTLV-III is not known to have in vitro transforming activity.
The diagnosis of HTLV-I infection is usually based on serum antibody response to HTLV-I peptide antigens. This usually involves an initial screening assay to identify HTLV-I antibodies, based on an enzyme immunoassay (EIA) with HTLV-I virion peptides. The assays presently used for blood screening detect about 0.5 to 0.05% HTLV-I and HTLV-II positives in blood donors in the United States; of these about 4 out of 5 are false positives. Therefore, positive sera must be further tested in a confirmatory assay, using Western blotted HTLV-I viral lysate. Current blood testing procedures require that individuals possess antibodies to both the HTLV-I p24 gag protein and at least one of the envelop proteins gp46, and gp68 (public health service working group). However, it has proven to be technically difficult to detect gp46 or gp68 proteins using a Western blot assay. Therefore a second round of confirmatory radioimmunoprecipitation assays must often be performed to detect antibody reaction to the HTLV-I envelope proteins.
A partial solution to this problem was provided by the molecular cloning of a 134 amino acid portion of the transmembrane glycoprotein gp21 (Samuel et al.). The recombinant protein, referred to as p21E protein, is reactive with sera from both HTLV-I and HTLV-II infected individuals, and has been successfully incorporated into Western blot assays for confirmation of HTLV infection (Lillehoj et al., Lipka et al. 1991). However, the p21E protein was also found to be reactive with 0.6% of HTLV negative blood donors (Lal, et al.). In addition, much higher rates of reactivity to p21E (approximately 5% in U.S. blood donors) are observed in individuals who are reactive in HTLV screening EIA tests, but who do not Possess antibodies to both HTLV-I gag and env gene products when tested by HTLV confirmatory assays and thus do not meet established criteria for being HTLV infected (Lal, et al.; Lipka, et al., 1991). Additionally in the study by Lipka et al. (Lipka et al. 1991) all of the p21E reactive-HTLV indeterminate individuals were negative for the presence of HTLV-I and HTLV-II nucleic acids when tested by PCR using HTLV-I and HTLV-II specific primers and probes. Therefore some individuals who are not infected with HTLV-I or HTLV-II possess antibodies which react with the p21E antigen. This fact has limited the use of the p21E recombinant protein, particularly in HTLV screening assays in which a high rate of false positives with HTLV-negative sera would result in the needless disposal of donated blood.
It would therefore be desirable to provide an improved method for detecting HTLV-I and HTLV-II positive sera. In particular, the improved test should be capable of detecting all HTLV-I and HTLV-II positive sera, with a minimum number of false positives, and also be able to distinguish HTLV-I from HTLV-II infected sera.